Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Expression of ß(2) adrenoreceptors on peripheral blood mononuclear cells in patients with primary and secondary progressive multiple sclerosis: a longitudinal six month study

Background: ß(2) Adrenoreceptor expression on peripheral blood mononuclear cells is increased in progressive multiple sclerosis. This increase has been correlated with disease activity in relapsing-remitting multiple sclerosis. Objective: To determine the ß(2) adrenoreceptor expression in primary and secondary progressive multiple sclerosis in relation to findings on magnetic resonance imaging (MRI) and clinical disease activity. Methods: 10 patients with multiple sclerosis were studied (five with primary progressive and five with secondary progressive forms of the disease) over a period of six months. Monthly clinical and MRI assessments of the brain and spinal cord were carried out. ß(2) Adrenoreceptor expression was assessed monthly using a ligand binding assay with [(125)I]iodocyanopindolol. Expression of ß(2) adrenoceptors on peripheral blood mononuclear cells was also assessed in five normal controls over a similar period. Results: The mean (SEM) value of ß(2) adrenoreceptor density for the five normal controls was 1346 (183) sites/cell, with affinity Kd of 120 (40) pM. MRI disease activity in primary progressive multiple sclerosis was reported on two occasions and on those occasions the expression of ß(2) adrenoreceptors was increased in excess of 1900 sites/cell; in the remaining 28 observations ß(2) adrenoreceptor expression was within the normal range (800 to 1900 sites/cell). In patients with secondary progressive disease, MRI disease activity was observed on 16 occasions. In these patients expression of ß(2) adrenoreceptors was increased in excess of 2000 sites/cell in all measurements except in one subject who did not show MRI activity throughout the six months period of study. The affinity of the receptors was within the normal range in all cases. Conclusions: Increased expression of ß(2) adrenoreceptors was correlated with MRI disease activity in two patients with primary progressive multiple sclerosis. In secondary progressive multiple sclerosis, increased expression of ß(2) adrenoreceptors tended not to correlate with MRI disease activity. This may reflect a persistent Th1 immune reaction in the secondary progressive form of the disease

Expression of βadrenoceptors on circulating mononuclear cells in hypertensives and normotensives before and after reduction of central sympathetic outflow by clonidine

We have studied βadrenoceptor number and affinity on peripheral blood mononuclear cells (PBMCs) in normotensives (NT) and hypertensives (HT), before and after intravenous administration of clonidine, an α2-adrenoceptor agonist which lowers blood pressure predominantly by reducing central nervous system sympathetic outflow. After clonidine, there was a decrease in blood pressure and plasma noradrenaline (NA) and adrenaline (Ad) levels, with an increase in growth hormone (GH) levels, in both NT and HT. There was no difference in basal βadrenoceptor densities on PBMCs between NT and HT. After clonidine at 30 and 60 min, there was an increase in βadrenoceptor density associated with a low affinity in NT. In HT, no changes were observed. The increased βadrenoceptor densities on PBMCs in NT after clonidine, returned to baseline values after 2h. Short term up-regulation of βadrenoceptors on PBMCs in NT after clonidine is accompanied by a fall in blood pressure (BP) and plasma levels of catecholamines. The changes may represent a compensatory mechanism reflecting a rapid externalization-activation of adrenoceptors residing on the internal surface of the membranes with a change of the coupling ability between the receptor and the catalytic component. In HT, although the haemodynamic and neurohormonal response to clonidine was similar to NT, short term up-regulation of receptors did not occur. The lack of such response may mirror a form of regulatory dysfunction of βadrenoceptors in HT.</p